利用候選基因法及微衛星標幟法進行努比亞和臺灣黑山羊生長性狀及體型測量值之分子標幟開發

Abstract

在畜牧產業中，動物的生長性能表現向來是育種過程所高度重視的。由於生長性狀容易受到遺傳、性別、環境…等因子影響，而隨著分子生物學和生物技術的發展，透過分子標幟輔助選拔可以較為準確且更有效率的達到選拔目標。一般而言，尋找和驗證與欲改良性狀有關的分子遺傳標幟，是建立分子標幟輔助選拔的第一步，亦是最關鍵的步驟。本詴驗選擇兩候選基因─生長激素基因（growth hormone gene）和肌肉生長抑制素基因（myostatin gene），期望藉由候選基因及微衛星標幟基因多型性與羊隻生長性能表現之相關性分析，找出對於羊隻生長性能具有顯著影響之分子遺傳標幟。 本詴驗共分析 109 頭努比亞山羊和 32 頭臺灣黑山羊之生長激素基因、肌肉生長抑制素基因及 15 組微衛星標幟基因多型性，另蒐集 109 頭努比亞山羊之生長性狀及體型測量值，包含出生重、三月齡（離乳）與六月齡體重、體長、體高、胸圍及各階段的帄均日增重，以進行基因多型性與生長性能關聯性探討。詴驗結果顯示，使用直接定序法與 PCR-RFLP 方法分析，於上述兩個候選基因中共檢測到 24 個變異位點，其中有 6 個變異點位於生長激素基因第三外顯子；5 個變異點位於生長激素基因第四外顯子；3 個變異點位於生長激素基因第五外顯子；9 個變異點位於肌肉生長抑制素基因啟動子區域，而於肌肉生長抑制素基因 5’ 非轉譯區則檢測到五個核苷酸的缺失，某些變異位點具有較低的多型性，有的位點則不具多型性。此外，於努比亞和臺灣黑山羊肌肉生長抑制素基因啟動子區域中各檢測到 15 種和 7 種基因型組合，初步完成此區域之單套型分析顯示，分別於此兩族群中檢測到 7 種與 6 種不同之單套型。進一步將基因多型性與努比亞山羊生長性狀及體型測量值進行相關性分析，結果發現於生長激素基因第三外顯子第 99 位點處，僅能檢測到 TT 和 TC 兩種基因型，相較於帶有 TT 基因型羊隻，帶有 TC 基因型羊隻具有顯著較高之三月齡體重、六月齡體重、三月齡體長、離乳前帄均日增重以及出生至六月齡帄均日增重（P<0.05）。另採用交替基因替代模型估算肌肉生長抑制素基因啟動子區域單套型對於生長性狀及體型測量值的效應，結果顯示單套型 1 對於努比亞山羊三月齡體長具有顯著正面影響，單套型 2 與 3 對於努比亞山羊三月齡體高則具有顯著負面影響（P<0.05）。另一方面，使用相同的模型獨立估測各組微衛星標幟交替基因對於生長性狀及體型測量值之效應，於所檢視的 15 組微衛星標幟中，發現共有 13 組微衛星標幟的交替基因對於至少一種生長性狀表現具有顯著影響（P<0.05）。 綜合上述結果，本詴驗利用候選基因法及微衛星標幟法，開發出對於努比亞山羊生長性能具有顯著效應的分子遺傳標幟，希望未來能應用於山羊分子標幟輔助選拔系統中，以增進臺灣羊隻的生長性能表現。於各種不同的育種計劃內，期望透過選育帶有正向效應之交替基因、單套型，或是基因型組合的羊隻，能夠讓養羊生產者有機會提高羊隻生長性能表現之遺傳增益效果。

In livestock industry, growth performance of animal is always of great concern during breeding. The growth performance is subject to significant confounding by genetics, sex, environmental variables and so on. With the development of molecular biology and biotechnology, more accurate and efficient selection goal could be achieved by marker-assisted selection (MAS). Generally speaking, seeking and validating the molecular genetic markers of interested traits are the initial and crucial step to establish a MAS system. In this study, we assigned growth hormone gene (GH gene) and myostatin gene (MSTN gene) as candidate genes and hoped to find molecular genetic markers with significant effects on goat growth performance by association studies between goat growth performance and candidate gene as well as microsatellite marker polymorphisms. In this study, the polymorphisms and genotypes for caprine GH gene, MSTN gene and 15 microsatellite loci were determined in 109 Nubian and 32 Taiwan Black goats. Phenotypic data of Nubian goats including birth weight, 3 month (weaning) and 6 month body weight, body length, withers height, chest girth, and average daily gain (ADG) were recorded and calculated for association analysis. The results showed that total of 24 mutations were detected in GH and MSTN genes by sequencing and PCR-RFLP analyses: 6 in GH exon 3, 5 in GH exon 4, 3 in GH exon 5, 9 in MSTN promoter region, and 5 nucleotides deletion in MSTN 5’ untranslated region. The polymorphism per site examined is quiet low or just monomorphism on some sites. In MSTN promoter region, 15 and 7 genotype combinations were detected in Nubian and Taiwan Black goats, respectively. The haplotype analysis showed that 7 and 6 haplotypes were detected separately in these two populations. For growth traits and body conformation measurements tests, only two genotypes TT and TC could be detected on the site c.99 C→T in GH exon 3. GH c.99 TC individuals had significant higher 3 month weight, 6 month weight, 3 month body length, ADG before weaning, and ADG during birth to 6 month than GH c.99 TT individuals in Nubian goats (P<0.05). Allele substitution models were used to estimate associations between MSTN promoter region haplotypes and growth traits and body conformation measurements. The results indicated that haplotype 1 had significantly positive effect on 3 month body length while haplotype 2 and 3 had significantly negative effects on 3 month withers height in MSTN promoter region in Nubian goats (P<0.05). On the other hand, allele substitution models were also used to estimate the relationship between microsatellite marker alleles and growth traits and body conformation measurements. Thirteen out of the 15 microsatellites showed significant associations with at least one of the examined traits in Nubian goats (P<0.05). In conclusion, the molecular genetic markers searched by candidate gene and microsatellite marker approach in this study might be used in MAS system to improve growth performance of goat in Taiwan in the future. Selection of goat carrying the alleles, haplotypes or genotype combinations might offer breeders the chance to increase rates of genetic gain for growth performance under variety of breeding programs.